Down-regulation Of SCAP Inhibits Cardiomyocyte Damage Induced By High Glucose And Its Mechanism

ObjectiveTo study the effect of down-regulating the expression of sterol regulatory element binding protein cleavage-activating protein(SCAP)on the response of cardiomyocytes induced by high glucose and its possible mechanism.To lay the experimental foundation for exploring the regulation mechanism of SCAP on diabetic cardiomyopathy.MethodsCardiomyocytes(H9C2)were cultured in vitro and treated with high glucose(35m M)to establish a cardiomyocyte injury model.Then use Lipofectamine 3000 transfection reagent to transfer SCAP-si RNA and negative control si RNA into H9C2 cells treated with high glucose.The cells were divided into five groups: normal culture group(NG),high glucose group(HG),high glucose+negative control group(HG+NC),high glucose+si RNA1group(HG+si RNA1),high glucose+si RNA2 groups(HG+si RNA2).After that,the expression of SCAP protein was detected by western blotting and immunofluorescence.TUNEL was used to detect cell apoptosis.Western blotting detects the expression of apoptosis-related proteins(Cleaved Caspase-3,Bax and Bcl-2),NLRP3,Caspase-1,IL-1β,NF-κB p65 and p-NF-κB p65 proteins.Results1.Western blotting and immunofluorescence experiments showed that the expression of SCAP protein in H9C2 cells after high glucose treatment was significantly higher than that of the normal group(P<0.01).2.TUNEL experiment showed that in the H9C2 cells of the HG group,the apoptosis rate was significantly higher than the NG group(P<0.05).Western blotting results showed that in the HG group,the expression of Cleaved Caspase-3 protein and Bax/Bcl-2 were increased compared with the NG group(P<0.01).Western blotting results showed that by transfecting SCAP si RNA,the expression of SCAP protein was effectively down-regulated(P<0.01).TUNEL results showed that the apoptosis rate of cells in the HG + si RNA groups were significantly lower than that in the HG +NC group(P<0.01)and the expression of Cleaved Caspase-3 protein and Bax/Bcl-2 were reduced(P<0.05).3.Reactive oxygen species experiment results showed that the ROS level in the HG group was significantly higher than that in the NG group(P<0.01).While the HG + si RNA groups was significantly lower than the HG + NC group(P<0.01).4.Western blotting results showed that the expression of NLRP3,Caspase-1 and IL-1β proteins in the HG group were significantly higher than that in the NG group(P<0.05).While the expression of NLRP3,Caspase-1 and IL-1β proteins in the HG + si RNA groups were reduced compared with the HG+ NC group(P<0.05).5.Western blotting results showed that the expression of p-NF-κB p65 in the HG group was significantly higher than that in the NG group(P<0.05).While the expression p-NF-κB p65 in the HG + si RNA groups was reduced compared with the HG + NC group(P<0.05).ConclusionsThis experiment suggests that SCAP is closely related to the damage of diabetic cardiomyopathy.Down-regulating the expression of SCAP can inhibit the apoptosis,the level of ROS and inflammation of cardiomyocytes induced by high glucose.It suggests that SCAP may be involved in the damage of diabetic cardiomyopathy,and its mechanism may be related to the NLRP3 inflammation pathway and NF-κB signaling pathway.It is suggested that further exploration of the function of SCAP may be of great significance to the diagnosis and treatment of diabetic cardiomyopathy.